The Role of Microorganisms in the Formation of a Stalactite in Botovskaya Cave, Siberia – Paleoenvironmental Implications

Biogeosciences, 10, 6115–6130, 2013 Open Access www.biogeosciences.net/10/6115/2013/ doi:10.5194/bg-10-6115-2013 Biogeosciences © Author(s) 2013. CC Attribution 3.0 License.

The role of microorganisms in the formation of a stalactite in Botovskaya Cave, Siberia – paleoenvironmental implications

M. Pacton1,*, S. F. M. Breitenbach1, F. A. Lechleitner1, A. Vaks2, C. Rollion-Bard3, O. S. Gutareva4, A. V. Osintcev5, and C. Vasconcelos1 1Geological Institute, ETH Zurich, Sonneggstrasse 5, 8092 Zurich, Switzerland 2Department of Earth Sciences, University of Oxford, South Parks Road, Oxford OX1 3AN, UK 3Centre de Recherches Pétrographiques et Géochimiques, UMR 7358, Université de Lorraine, 54500 Vandoeuvre-lès-Nancy, France 4Institute of the Earth’s Crust, Russian Academy of Sciences, Siberian Branch, 128 Lermontova Street, Irkutsk 664033, Russia 5Arabica Speleological Club, Mamin-Sibiryak Street, P.O. Box 350, Irkutsk 554082, Russia *now at: Laboratoire de Géologie de Lyon : Terre, Planètes, Environnement Campus de la Doua, Universite Lyon 1 2 Rue Raphael Dubois, 69622 Villeurbanne Cedex, France

Correspondence to: M. Pacton ([email protected])

Received: 20 March 2013 – Published in Biogeosciences Discuss.: 8 April 2013 Revised: 25 July 2013 – Accepted: 15 August 2013 – Published: 27 September 2013

Abstract. Calcitic speleothems in caves can form through negative δ13C excursions. The microscale biogeochemical abiogenic or biogenic processes, or through a combina- processes imply that microbial activity has only negligible tion of both. Many issues conspire to make the assess- effects on the bulk δ13C signature in speleothems, which is ment of biogenicity difficult, especially when focusing on more strongly affected by CO2 degassing and the host rock old speleothem deposits. This study reports on a multiproxy signature. analysis of a Siberian stalactite, combining high-resolution microscopy, isotope geochemistry and microbially enhanced mineral precipitation laboratory experiments. The contact between growth layers in a stalactite ex- 1 Introduction hibits a biogenic isotopic signature; coupled with morphological evidence, this supports a microbial origin of calcite The growth of speleothems such as stalactites and stalag- crystals. SIMS δ13C data suggest that microbially mediated mites through the precipitation of calcite has commonly speleothem formation occurred repeatedly at short intervals been viewed as an abiogenic process (e.g., Kendall and before abiotic precipitation took over. The studied stalactite Broughton, 1978; Broughton, 1983a, b, c). However, there also contains iron and manganese oxides that have been me- is a growing body of research suggesting that microbes diated by microbial activity through extracellular polymeric may play an important role in carbonate precipitation during substance (EPS)-influenced organomineralization processes. speleothem growth (e.g., Jones and Motyka, 1987; Northup The latter reflect paleoenvironmental changes that occurred and Lavoie, 2001; Baskar et al., 2005, 2006; Mulec et al., more than 500 000 yr ago, possibly related to the presence of 2007; Jones, 2010). In caves, a variety of precipitation and a peat bog above the cave at that time. dissolution processes results in the deposition of carbon- Microbial activity can initiate calcite deposition in the ate speleothems, silicates, iron and manganese oxides, sul- aphotic zone of caves before inorganic precipitation of fur compounds, and nitrates, but also in the breakdown of speleothem carbonates. This study highlights the importance limestone host rock. Cave microbes mediate a wide range of of microbially induced fractionation that can result in large destructive and constructive processes that collectively can influence the growth of speleothems and their internal crystal

Published by Copernicus Publications on behalf of the European Geosciences Union. 6116 M. Pacton et al.: The role of microorganisms in the formation of a stalactite in Botovskaya Cave, Siberia fabric (Jones, 2010). Constructive processes include microbe calcification, trapping and binding by filamentous microbes, and/or mineral precipitation (Cañaveras et al., 2001; Jones, 2001). Destructive processes include microbially influenced corrosion or dissolution of mineral surfaces that can occur through mechanical attack, secretion of exoenzymes, organic and mineral acids (e.g., sulfuric acid), and a variety of other mechanisms (for a summary please refer to Sand, 1997). Of particular interest in cave dissolution processes are reactions involving iron-, sulfur-, and manganese-oxidizing bacteria (Northup and Lavoie, 2001). Iron oxides and hydroxides are most often observed as coatings or crusts and as powder on clastic cave walls, but they also exist as typical speleothems such as stalactites (e.g., Caldwell and Caldwell, 1980; Jones and Motyka, 1987). Several descriptive studies have established the association of bacteria with iron deposits in caves, but experimental evidence for an active microbial role in the formation of iron deposits in caves is still lacking (Northup and Lavoie, 2001). Fig. 1. Map of Siberia, showing the location of Botovskaya Cave. With regard to flora and fauna, caves are usually divided into two segments: (i) a twilight zone and (ii) an aphotic zone characterized by no light and few microbes (Jones, 2010). In 2 Study site this study we focus on a stalactite found in the aphotic zone. Botovskaya Cave is located in Siberia (55◦1705900 N, The sample contains mainly calcite and ferromanganese ox- 105◦1904600 E, 750 m above sea level) (Fig. 1 and Fig. S3 in ides, which is unusual in this continental setting as they are Vaks et al., 2013), and developed as a > 68 km-long hori- widely encountered in warm environments (e.g., Spilde et al., zontal maze of passages along tectonic fissures in a 6 to 12 m 2005). The presence of microbes does not automatically im- thick Lower Ordovician limestone layer which is sandwiched ply that they played a role in the formation of the surrounding between marine sandstone and argillite (Filippov, 2000). minerals, because they may simply have been buried dur- The depth of the cave is 40–130 m below the surface. Be- ing mineral precipitation (Polyak and Cokendolpher, 1992; yond a few meters from the small entrances, the light inten- Forti, 2001). Assessment of the exact role that the microbes sity level in the cave is zero. Its passages are characterized played in the mineral precipitation is usually considered im- by massive clay infillings. The cave is located in discontinu- possible to make (Jones, 2010). However, by combining mi- ous permafrost, as evident from massive cave ice bodies with croscopical and geochemical evidence at a high spatial res- < 0 ◦C temperatures in the western part of the cave. Micro- olution, we aim to constrain precisely the role of microbes climatic monitoring reveals that cave air temperature varies in stalactite formation in this cave. Calcite δ13C has been only slightly, from just below 0 ◦C in the cold zones to 1.6– interpreted as reflecting surface vegetation changes (C3 vs. 1.9 ◦C in its warmest parts (see Fig. S4 in Vaks et al., 2013). C4: Brook et al., 1990; Dorale et al., 1992; Bar-Matthews The eastern cave section is the only area where water seepage et al., 1997; Hou et al., 2003; Denniston et al., 2007), al- occurs and speleothems grow today (Vaks et al., 2013). Sam- though more recent studies point to cave air ventilation as a ple SB-p6915 was collected several hundred meters from the major influence (Tremaine et al., 2011). It has been demon- nearest entrance, in the wet and aphotic zone. strated that degassing of CO from the dripwater controls the 2 The region surrounding the cave is covered by taiga rate of calcite precipitation (Mickler et al., 2004; Spötl et al., forest and receives ca. 400 mm annual precipitation. 2005; Bourges et al., 2006; Baldini et al., 2008; Kowalczk Mean annual surface air temperature is −2.8 ◦C, ranging and Froelich, 2010), as well as the isotopic composition of from +35 to −40 ◦C. dripwater and subsequent calcite (Mattey et al., 2008; Müh- linghaus et al. 2007, 2009; Oster et al., 2010; Frisia et al., 2011; Lambert and Aharon, 2011). These studies show that – 3 Methods if carefully evaluated – δ13C can be used as a paleoclimatic indicator. However, the potential impact of microbial activity 3.1 Samples on carbon isotope fractionation is usually overlooked in paleoclimate studies. We aim to investigate this impact in detail For this study we use subsamples from stalactite SB-p6915, in the current work. which is asymetrically shaped and has a diameter of 3.5– 4 cm. The sample was found in 2001, more than 1 km deep inside Botovskaya Cave (Supplement Fig. 1), and consists

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3.2 Mineralogy

For X-ray diffraction analyses (XRD), samples were ground to a ﬁne powder in an agate mortar. Samples were deposited on a silicon wafer in a plastic sample holder. We employed a Bruker AXS D8 Advance instrument at ETH Zurich, equipped with a scintillation counter and automatic sampler rotating the sample.

3.3 Microscopy

The laminae observed in stalactite SB-p6915 were studied by scanning electron microscopy (SEM) on polished and platinum-coated thin sections, using a Zeiss Supra 50 VP SEM at the University of Zurich, Switzerland. Semi- quantitative elemental analyses of micron-sized spots were obtained using an energy dispersive X-ray spectrometer Fig. 2. Location of the drilling trench of the stalactite from (EDS; EDAX, University of Zurich, Switzerland) during Botovskaya Cave at Layer B (upper arrow), where the sample for U- SEM observations. Th dating was taken. Note the asymmetric distribution of ferromanganese oxides in the stalactite. At least 2 hiatuses (marked by arrows) divide between Layer B and Hiatus D-E, where the youngest 3.4 Isotope analysis microbial presence can be seen as a thin dark-colored layer. In situ, spatially highly resolved carbon and oxygen isotope composition was determined using a Cameca IMS 1270 ion of a ca. 15 mm-thick white calcite core, surrounded by asy- microprobe at the CRPG-CNRS, Nancy, France, following metric dark brown-black layers of a total thickness of 10– the methodology outlined in Rollion-Bard et al. (2007). A 16 mm, which then give place to another white calcite layer primary Cs+ beam of 10 nA intensity was focused on spot of ca. 10 mm thickness (Fig. 2). The change from the white size of ca. 20 µm. The normal incidence electron gun was core to the dark layers is abrupt, with a precursory event that used to compensate for sample charging during analysis. deposited a brown ring about 2 mm closer to the stalactite’s Measurements of carbon and oxygen isotope ratios were con- center. The dark layers are compact on one side, but are in- ducted in multicollection mode, using one off-axis Faraday tercalated with white calcite on the opposite side, where the cup (L02) and the central electron multiplier, and two off- dark rings fade until they are interrupted. The dark-colored axis Faraday cups (L02 and H1), respectively. A liquid nitro- crusts display corrosion surfaces, as they are characterized gen cold-trap was used to lower the gas pressure in the spec- by an irregular surface topography. The alternating white and imen chamber and then to ensure the stability of the mea- dark layers show asymmetric stalactite growth that is likely surements. The instrumental mass fractionations (IMF) were the result of preferential water flow down one side. An asym- determined for reference materials of calcite, dolomite and metric growth due to cave wind is unlikely because multiple magnesite to take the effect of the magnesium content on expeditions at different seasons never observed any cave air the IMF into account for oxygen isotope measurements, as current in this (as well as many other) sections of Botovskaya described in Rollion-Bard and Marin-Carbonne (2011). No Cave. The latter is most likely due to the location of the sta- such effect was detected for carbon isotope analyses. The lactite more than 1 km from the nearest opening and in elim- typical acquisition time was 3 s during 40 cycles for carbon inating any possibility of significant air flows (Supplement isotope compositions and 25 cycles for δ18O analyses. Prior Fig. 1). The shallow passages also prohibit the development to each analysis, the following automated procedure was per- of vertical airflow, as passages most often have dimensions formed: (1) secondary ion beam centering within the field of 30–100 cm in width and 40–200 cm in height. aperture by adjusting the transfer lens deflector voltages, and Dripwater was collected in February 2011 close to stalac- (2) magnetic field scanning and peak centering. This proce- tite SB-p6915 in Botovskaya Cave. The samples were col- dure leads to an internal precision (2σ) of better than 0.2 ‰ lected using sterilized glass bottles and plastic tubes, and for δ13C and an external reproducibility (1σ) of ca. 0.5 ‰, were stored refrigerated and maintained at 4 ◦C until ship- based on repeated analyses of reference materials, as well as ping to ETH Zurich. an internal precision of better than 0.1 ‰ and an external re- producibilty of ≈ 0.3 ‰ for δ18O. For comparison, 102 carbonate samples were milled across the same sampling trench using a digitally controlled micromill (Sherline®) at 50 µm increments parallel to the www.biogeosciences.net/10/6115/2013/ Biogeosciences, 10, 6115–6130, 2013 6118 M. Pacton et al.: The role of microorganisms in the formation of a stalactite in Botovskaya Cave, Siberia

Table 1. U-Th dating results for the SB-6915-B stalactite layer (Vaks et al., 2013). The corrected values are shown in the 3rd and 4th rows.

Sample 238U (ppm) 232Th (ppb) (230Th/232Th) (232Th/238U) 2σ abs (230Th/238U) 2σ abs (234U/238U) 2σ abs Raw Age (ka BP) 2σ 1.00023 0.00304 1.00772 0.00278 496 60 (230Th/238U) corr. 2σ abs (234U/238U) corr. 2σ abs Corr. Age (ka BP) 2σ SB-p6915-B 3.51 1.02 10241 9.54 × 10−5 9.15 × 10−7 1.00023 0.00304 1.00772 0.00278 496 61 growth axis (perpendicular to the crack observed in Fig. 8). uate the role that EPS may play in precipitating iron oxides The milled powder was measured on a Gasbench II coupled under controlled laboratory conditions. The liquid medium to a Delta V Plus (Thermo Fisher Scientific) mass spectrome- was prepared as follows: 0.5 g Fe(NH4)2(SO4)2 × 6H2O and ter at ETH Zurich. Analytical details can be found in Breiten- 10 mg FeCl3 × 6H2O were added to one liter of 0.2 µm fil- bach and Bernasconi (2011). The external standard deviation tered dripwater from the stalactite. Erlenmeyer flasks con- (1) for both isotopes was better than 0.07 ‰. All values are taining 100 mL of the liquid were inoculated with biofilms expressed in permil and referenced according to the Vienna (mm-scale thickness). Abiotic “blank” samples containing Pee Dee Belemnite (VPDB) standard. only the liquid medium were prepared as controls. Plastic push caps were placed on top of each test tube to prevent 3.5 Dating contamination and minimize evaporation. Cultures and controls were incubated at room temperature Stalactite SB-p6915 has been dated at Oxford University us- in the presence of sunlight for three weeks. Biofilms were ex- ing the U-Th method (Vaks et al., 2013). For U-Th dating, amined using transmitted light microscopy to determine the 132 mg of calcite were taken from the outer rim (Layer B presence of precipitates. Samples of biofilms and secondary in Fig. 2). Details of the dating procedure are given in the mineral precipitates were collected using a sterile spatula and Supplementary Online Materials of Vaks et al. (2013). examined using SEM. Precipitates were collected and analyzed for their mineralogical composition using XRD. 3.6 Laboratory iron-oxide precipitation experiments

Biofilms were cultured from water samples collected in the cave. The biofilms had been produced from a microbial mat 4 Results from Lagoa Vermelha, Brazil (Vasconcelos et al., 1995) under stress-controlled conditions, i.e., hypersalinity, in order to 4.1 Dating results and approximate age of produce a significant amount of extracellular polymeric sub- microbial deposition stances (EPS). Prior to the iron experiments, the biofilm was analyzed using SEM, TEM (embedding in Epoxy and cut The U-Th age of Layer B in stalactite SB-6915 has been de- into ultrathin sections) and XRD in order to validate the ab- termined as 496 ± 61 ka BP (before present, i.e., 1950 AD) sence of any mineral phases (carbonates, iron oxides, amor- (Fig. 2, Table 1). According to Vaks et al. (2013), Quater- phous Mg-Si phases, etc.) and the abundance of microbes. nary speleothem growth in Botovskaya Cave occurred only Transmission electron microscopy data indicate a complete during the warmest episodes of interglacial periods when absence of permineralization within EPS and very few bac- permafrost above the cave thawed. The dated layer corre- teria. The abundance of EPS over isolated microorganisms sponds to interglacial marine isotope stage (MIS) 13. If the is supported by DAPI-staining (very few fluorescent bac- speleothem growth during periods older than 500 ka fol- teria) and Gram coloration, suggesting that photosynthetic lowed the same pattern as during the younger periods, the organisms were unlikely to have played a role in mineral calcite layers represent interglacial speleothem deposition, formation. Moreover, TEM examinations of bacteria show whereas thin white and brown layers represent hiatuses, i.e., cell disruptions with loss of intracellular materials, which interruptions in speleothem deposition associated with colder would suggest that they are dying (e.g., Diaz-Visurraga et permafrost conditions (Vaks et al., 2013). Each hiatus thus al., 2010). Based on the TEM and DAPI-staining results, we represents a surface of the stalactite during a period of growth exclude light-favored metabolically driven iron oxide for- interruption (most likely permafrost conditions). The hiatus mation. Although the composition and quantity of the EPS between Layers E and D is the last speleothem surface on vary depending on the type of microorganism and the differ- which a microbial community was observed. Two calcite lay- ent environmental conditions under which the biofilms are ers and two hiatuses separate Layer B and the last period cultured, this does not alter their ability to bind metal ions where microbial occurrence was observed (Fig. 2), suggest- from solutions (Decho, 1990; Santschi et al., 1998). Biofilms ing that it occurred at least two glacial-interglacial cycles be- were cultured from dripwater samples in 125 ml Erlenmeyer fore MIS-13. Although only tentative, this line of argument flasks at room temperature (∼ 20 ◦C) and in the presence of may place the minimum age of the end of microbial growth sunlight. Precipitation experiments were performed to eval- on the stalactite surface at the beginning of interglacial MIS-

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a b

c d

e f c

Figure 3, Pacton et al. Fig. 3. Secondary electron images of the surface of the stalactite: (a, c) the contacts between layers show the terminations of columnar crystals characterized by cavities and irregular crystals; (d, f) irregular small calcite crystals are covered by EPS-like structures showing sheet morphologies (arrows).

17, about 700 ka BP. Detailed U-Pb dating could possibly reﬁne this provisional chronology.

4.2 Texture and morphology of the stalactite

4.2.1 White laminae

The stalactite is characterized by an alternation of thickly laminated columnar crystals and thinner layers. Trigonal prismatic calcite crystals, forming columnar horizons up to 1 mm long and 0.2 mm wide, are widely distributed. The contacts between layers are characterized by cavities and irregular crystals at the terminations of the columnar crys- Fig. 4. Elemental analyses using EDAX in the SEM area (Fig. 9f): (a) organic composition of the biofilm as shown by the high C peak; tals (Fig. 3a–d). The cavities are made by small calcite (b) surrounding calcite; (c) biofilm is locally permineralized as Mg- crystals, which are covered by EPS-like structures showing Si-O. sheet morphologies (Fig. 3e, f). Elemental analyses confirm the organic composition, shown by the higher carbon peak (Fig. 4a), compared with that of the surrounding calcite crystals (Fig. 4b). This biofilm is locally composed of Mg and by the occurrence of filaments (Fig. 6c) and EPS (Fig. 6d). Si (Fig. 4c). Similar microbial structures on the calcite have Smaller rhombs are locally covered by a thin biofilm made been observed in other speleothems from the same cave. of low-magnesium calcite (Fig. 6e, f). Close to columnar calcite, smaller calcite rhombs exhibit dis- The carbonates are identified as calcite by XRD spec- solution features (Fig. 5a–d). The latter are sometimes cov- tra (Fig. 7a). SEM and EDX analyses confirm the presence ered by aluminosilicates (Fig. 5e, f). Microbes are associ- of this mineral and also reveal the presence of amorphous ated with these smaller rhombs (Fig. 6a–d) and are confirmed Mg-Si (amMg-Si) phases that were not detected by XRD www.biogeosciences.net/10/6115/2013/ Biogeosciences, 10, 6115–6130, 2013 6120 M. Pacton et al.: The role of microorganisms in the formation of a stalactite in Botovskaya Cave, Siberia

a b

c d

e f

Fig. 5. Secondary electron images of the surface of the stalactite: Figure 6, Pacton et al. (a–c) dissolution features (arrows) of the small calcite rhombs close Fig. 6. Secondary electron images of the calcite rhombs in the cav- to columnar calcite; (d) they are sometimes covered by a film; (e, f) ities: (a–d) microbes are widely distributed as filaments (black ar- elemental analyses of this film show that it is composed of alumi- row) and EPS (white arrows). (e) Smaller rhoms are locally covered nosilicates on the top of calcite rhombs. by a thin biofilm; (f) elemental analysis of the biofilm showing that it is made of low-magnesium calcite. analysis. The thickly laminated, columnar calcite crystals 4.2.2 Dark-brown crusts have SIMS δ13C values between −1.27 ‰ and +1.06 ‰, which are in the same range as obtained by the isotope ra- Stalactite SB-p6915 shows multiple dark brown to black − + tio mass spectrometry (IRMS) ( 2.3 ‰ to 2.2 ‰), and ob- crusts (Fig. 2). XRD analyses detected only calcite, which served in several other stalagmites from Botovskaya Cave is poorly defined in the diffractogram, indicating that all the (Supplement Table 1, Breitenbach, 2004). The contacts at crusts had a low crystallinity (Fig. 7b). the different cracks, which are characterized by thinner lay- 13 SEM analyses reveal an abundance of predominantly ers, display very negative SIMS δ C values ranging from spherical bodies with sizes ranging from a few microns to − − 5.27 to 13.19 ‰ (Fig. 8 and Supplement Figs. 2–3). 20 µm (Fig. 10a–d). These are composed of iron oxides that This strong depletion is not found in the IRMS data, where cover depressions between calcite crystals (Fig. 10a) and can 13 − δ C values remain in the range from 0 to 2 ‰ (Fig. 9). be associated with Mg-Si phases (Fig. 10). They are com- The crack however is visible as carbon and oxygen shifts 13 posed of acicular needles that are radially arranged, thus towards more negative values. The IRMS δ C decrease forming coccoid bodies (Fig. 10d, e). These bodies display a has to be relativized, since shifts of this magnitude occur close association with EPS, as the needles are located within throughout the analyzed section. 18 or below EPS (Fig. 10c–e). SIMS δ O also reflects a ca. three permil depletion of Microborings are present on the surface of calcite crystals. −11.9 to −15 ‰ around the crack, while this is less sig- 18 These are characterized by open borings and resin casts: open nificant in the IRMS δ O data: here less depleted (1.5 to borings consist of solitary spherical cells of an average diam- 2 ‰ compared to before the crack) values are observed. Over eter of 1–10µm (Fig. 11a, b). Cement-filled microborings are 18 − − the entire IRMS profile, δ O varies from 12 to 14.5 ‰ occasionally visible, and consist of Fe-Si phases and low- (Fig. 9). magnesium calcite cast precipitates (Fig. 11c, d). The microborings are covered by Fe-Si-Mn crusts composed of nanofilaments, which form flakes (Fig. 11e, f). Close to depressions, microborings are corroded by iron oxides (Fig. 12a, b). The latter are characterized by fibrous radial structures, which are

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Fig. 7. XRD analyses of various zones of stalactite show that (a) the thick and thin layers consist mainly of calcite, and that (b) dark-brown crusts are amorphous as calcite is the only mineral present in the diffractogramm. intermixed with EPS (Fig. 12c). The contact between calcite the stalactite. Rare nanoparticles of manganese oxides are crystal and these crusts shows corrosion features (Fig. 12d). present, showing a crenulated surface structure (Fig. 14d). Where this cover is thin, microborings are still visible under randomly distributed nanofilaments (Fig. 12e, f). At the inter- 4.3 Laboratory iron-oxide precipitation experiments face between calcite and oxy-hydroxides, partial dissolution of calcite rhomboedra is observed (Fig. 13). These rhombs We designed a laboratory experiment to compare abiotic and are characterized by irregular outlines (Fig. 13b–d) and show biotic iron-oxide precipitation under controlled conditions, a close relationship with microorganisms. Filaments are lo- and to allow the characterization of newly formed precipi- cated on top of calcite crystals and part of iron oxides, sug- tates. After being inoculated with the iron-rich medium, the gesting that they developed contemporaneously (Fig. 13c, d). biofilms are found encrusted with dark-brown mineral pre- Unlike spherical microborings, other cavities consist cipitates when viewed using phase-contrast light microscopy. of tunnels that are locally covered by an alveolar low- Examination using SEM reveals rosettes with minor amounts magnesium calcite network (Fig. 14). This network is of flakey globular aggregates and with a crenulated surface in magnesium-enriched compared to the calcite that forms the biofilm that formed from the dripwater (Fig. 15a). EDS analyses of these precipitates show that they contain Fe, C, www.biogeosciences.net/10/6115/2013/ Biogeosciences, 10, 6115–6130, 2013 6122 M. Pacton et al.: The role of microorganisms in the formation of a stalactite in Botovskaya Cave, Siberia

δ18O (‰) δ13C (‰) A -16 -12 -8 -4 0 B

100 μm 100 μm

Figure 8, Pacton et al. Fig. 8. Microphotographs of segments of stalactite sample after ion- probing. δ13C measurements indicate more negative values in the cavities, as shown by the SEM picture. and O, thus identifying them as iron oxides. XRD analysis Fig. 9. Proﬁle of δ13C and δ18O variations with depth in stalactite of these precipitates veriﬁed them to be amorphous. While 2-N obtained by micro-drilling and conventional acid dissolution iron-oxide crystal aggregates are present in the abiotic cave analysis. The grey area corresponds to a crack. water control, no rosettes have been observed (Fig. 15b).

5 Discussion permineralized amMg-Si phases (Sanz-Montero et al., 2008; Pacton et al., 2012). Many of the biofilms found throughout 5.1 Role of microbes in carbonate precipitation the stalactite commonly cover the surfaces of block-like and weathering calcite crystals. Therefore, biofilms seem to help initiate layer formation on the stalactite via organomineralization Potentially, microbes can influence the growth of processes characterized by small calcite crystals. Subse- speleothems by microbe mineralization, by trapping quently, abiotic calcite precipitation leads to the formation of and binding detrital grains on the substrate, and/or by thicker columnar calcite crystals. The latter may be further mediating mineral precipitation (Léveillé et al., 2000a, b; colonized by endolithic microborers, as suggested by the Cañaveras et al., 2001; Jones, 2001, 2010). In our sample, coccoid shapes in the studied sample. These could be similar the thickly laminated columnar calcite crystals are enriched to endolithic microborings produced by algae and some in 13C (with δ13C values of ∼ 0 ‰) compared to small cyanobacteria (Harris et al., 1979). Despite the fact that only calcite crystals (ca. −6 to −13 ‰ using SIMS), whereas a few taxa are found in the twilight zone of the cave, these no significant variations are observed in the SIMS δ18O microborers may be Geitleria calcarea, Loriella osteophila values (Fig. 8). Several δ13C and δ18O SIMS profiles along or other bacteria (Jones, 2010). While there is no evidence with SEM including porous cracks confirm that the 13C of trapping and binding or filament encrustation, this study depletion is typically associated with the porous cracks, gives further evidence of microbes indirectly influencing which are characterized by small low-magnesium calcite precipitation in a Pleistocene stalactite. rhombs and local microbial filaments. More negative δ13C isotope signatures can result from microbial fractionation of 5.2 Role of microbes in ferromanganese mineral C (Melim et al., 2001; Cacchio et al., 2004; Léveillé et al., formation 2007). The preferential uptake of 12C-enriched compounds during microbial mineralization leads to 13C-depletion in the The amorphous nature of ferromanganese oxides (docu- precipitated carbonate compared to the source material (e.g., mented by XRD) is the initial form of metal precipitated in Lee et al., 1987; Pacton et al., 2012). More specifically, the caves and has been proposed as a typical feature of microbial observed carbon isotope depletion coincides with the loca- precipitates (Northup and Lavoie, 2001; Spilde et al., 2005; tion of the mineralized biofilm that served as the template for Tebo et al., 1997). The globular and rosette arrangement of calcite precipitation. These unusual mineral morphologies, these oxides is however unexpected, unlike the wide range coupled with a high Mg2+ content (Fig. 4) are evidence of morphologies described in the literature, e.g., bacterial fil- for EPS-influenced organomineralization (Dupraz et al., amentous and coccoid bodies, or sheet-like minerals (e.g., 2004). Similar small magnesium calcite crystals have been Chafetz et al., 1998). recently observed in speleothem associated with microbial Several researchers invoked metabolic precipitation mech- activity, suggesting a possible biomineralization process anisms such as chemolithotrophy to trigger iron and/or (Frisia et al., 2012). Analogous to freshwater microbial manganese oxide formation (Peck, 1986; Northup, 2003; carbonates, precipitation appears to be initiated from EPS as Northup et al., 2000; Spilde et al., 2005). Fortin and

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Fig. 10. Secondary electron images of the dark-brown crusts: (a) Fig. 11. Secondary electron images of the surface of the stalactite: iron oxides are located in the depressions within calcite crystals; (a) a thin layer cover calcite surface that includes microborings (ar- (b, c) globular structures with a size ranging from a few microns to rows). They consist of solitary cells that are spherical in shape and 20 |mum; (d, e) spherical bodies are composed of acicular needles average 1 to 10 µm in diameter; (b) microborings include open bor- that have a radial arrangement. They display a close association with ings (1) and resin casts (2); (c, d) elemental analyses indicate that EPS (arrows), as the needles are within or below EPS; (f) elemental open borings are made of low-magnesium calcite, whereas resin analysis showing that Fe and O are the main components of these casts are made of Fe, O and Si; (e) the thin layer-covering calcite crusts and are associated with a small contribution of Mg and Si. crystals are composed of nanoﬁlaments forming ﬂakes; (f) elemental analyses of this layer reveal that it is made of Fe, Si and Mn.

Ferris (1998) discuss the capability of bacteria to provide nu- romanganese deposits in caves are usually considered an cleation sites favorable for iron and manganese deposition. end-product of microbially assisted dissolution and leach- Our laboratory experiments confirm the microbial origin ing of the underlying host carbonate, and of the enrich- of the Fe and Mn deposits in the Siberian stalactite. Although ment of iron and manganese through microbial oxidation mineral precipitation occurred as mineral crusts within both (Northrup et al., 2000, 2003; Boston et al., 2001). Ferric abiotic and biotic systems, rosettes similar to those present in iron is essentially insoluble at neutral pH, and amorphous the stalactite sample SB-p6915 only form in the biotic sys- Fe(III) oxide is the predominant form of Fe(III) reduced in tem. Apparently, biofilms allow iron oxide formation with these environments (Murray, 1979; Schwertmann and Tay- a different morphology than the abiotic system. These find- lor, 1977). Fe(II) and Mn(II) may have been released into the ings demonstrate that EPS promote rosette iron oxide forma- cave environment by iron- and manganese-oxidizing bacteria tion without the requirement of any microbial metabolism, (Northup et al., 2000). i.e., a passive mineralization of EPS. This suggests an EPS- Biotic oxidation of metals can occur either indirectly or di- influenced organomineralization process (as opposed to in- rectly. Indirect oxidation results from the release of oxidants, duced, in which a microbial metabolism can change, e.g., acids, or bases into the environment surrounding the micro- the alkalinity) (e.g., Dupraz et al., 2009). This process may bial cell, and leads to a change in redox conditions in the have been rather fast, as microbially accelerated Fe precipita- surrounding microenvironment (for a review see Tebo et al., tion rates are more likely related to exopolysaccharides and 1997). Direct oxidation may occur through the binding of ion microbial surface properties than to metabolic precipitation metal to negatively charged substances on the bacterial cell mechanisms (Kasama and Murakami, 2001). surface, or through the action of metal-binding proteins that The microbial community plays an active role in both are both intra- and extracellular (Ghiorse, 1984). the breakdown of bedrock and speleothem formation. Fer- www.biogeosciences.net/10/6115/2013/ Biogeosciences, 10, 6115–6130, 2013 6124 M. Pacton et al.: The role of microorganisms in the formation of a stalactite in Botovskaya Cave, Siberia

a b

c d

e f Fig. 13. Secondary electron images of the relationships between dark-brown crusts and calcite crystals: (a, b) partial dissolution of calcite rhomboedra as shown by the porous texture (black square); (c, d) irregular outlines of calcite rhombs (white arrows) closely associated with microorganisms (black arrows).

Figure 12, Pacton et al. which typically has a δ13C value near zero (e.g., Johnson Fig. 12. Secondary electron images of the corrosion features: (a, et al., 2006; Cruz et al., 2006; Cacchio et al., 2004). Long- b) calcite crystals corroded by iron oxides close to depressions, as term changes in speleothem δ13C values can reflect various shown by irregular outlines; (c) iron oxides characterized by fibrous changes in the carbon budget, such as shifts in the type of radial structures (arrows), which are intermixed with EPS (dashed vegetation overlying the cave (Denniston et al., 1999), de- arrows); (d) the contact between calcite crystal and these crusts shows corrosion features (arrow); (e) where this cover is thin, mi- gassing changes associated with high or low drip rates (Müh- croborings are still visible (arrows); (f) iron oxides are composed of linghaus et al., 2007), or variations in host rock dissolution. randomly arranged nanofilaments. Lower drip rates and prolonged CO2 degassing as well as a change from taiga forest to open tundra during times of massive drying would all result in enriched δ13C values. Major The observed layers, rich in ferromanganese oxides, sug- alterations of vadose fluid pathways or changes in dripwa- gest periods of intensified weathering and/or erosion above ter δ13C composition are also unlikely, and thus we argue the cave caused by humid (and relatively warmer, because of that more negative δ13C values are most likely the result of permafrost absence) conditions. Deposition of stalactite car- microbially mediated processes. The strong carbon isotope bonate (and the crusts) was only possible during warm inter- depletion within the crack (Fig. 9) found by SIMS analysis, vals when permafrost was absent above the cave. Contrary to along with EPS-like morphological evidence, further support manganese stromatolites found in caves (Rossi et al., 2010), a microbial origin. the deposits found in our study were essentially formed at the However, would microbial activity have a significant in- speleothem-air interface (e.g., Spilde et al., 2005). fluence on conventional (bulk) IRMS isotope analysis? The observed SIMS δ13C decrease is less clearly reflected in the 5.3 δ13C and δ18O as paleoclimate proxies IRMS stable isotope profile. The microbial mineralization in only very thin layers on the sample likely results in an atten- Since δ13C and δ18O ratios are routinely used as paleocli- uated IRMS δ13C decrease observed in bulk carbonate anal- mate proxies, we investigate if microbial activity might have ysis (−2 ‰ compared to −6 to −13 ‰ in the SIMS sam- a significant influence on the isotopic composition of the car- ples). The IRMS δ13C variability found throughout the ana- bonate, thus complicating the interpretation of isotope time lyzed section (unrelated to the crack) also suggests that the series in terms of climatic variations. microbial signature is of negligible importance for the over- Carbon in speleothem calcite has three main sources: (1) all isotope profile. We are positive that the studied IRMS 13 CO2 produced in the soil by respiration of organic material, δ C tracks record mainly environmental changes, and that with δ13C values ranging from −15 to −25 ‰; (2) C from the microbial overprint is very small, owing to the scale dif- 13 atmospheric CO2, normally with δ C values close to −7 ‰; ferences when comparing the ultra-high resolution SIMS and and (3) inorganic carbon from dissolved carbonate host rock, bulk IRMS carbonate analyses.

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Fig. 15. Secondary electron images of the ferromanganese crusts: (a) the bioﬁlm inoculated in the iron-rich medium. Note that 1 µm- wide rosettes showing a crenulated surface (black arrows) are closely related to extracellular polymeric substances (white arrows). (b) Detail of amorphous iron oxides in the sterile iron-rich medium appearing as nanoglobules. (c) Elemental analyses of the rosettes Fig. 14. Secondary electron images of the ferromanganese crusts (a) indicating a strong contribution of magnesium in the iron ox- covering microborings in calcite: (a) microborings made of tunnels; ides. (b, c) part of tunnels covered by an alveolar network; (d) particule displaying a crenulated surface structure; (e) elemental analysis of the alveolar network mainly composed of low-magnesium calcite; the role of microbes in speleothem formation related to hia- (f) elemental analysis of the manganese oxide particle. tuses and/or carbonate disruptions.

5.4 The host rock as potential source of iron The δ18O profiles found by both methods, SIMS and and manganese IRMS, can be explained by abiotic processes. The IRMS oxygen isotope profile shows a prominent shift from −12.5 One source of iron or manganese could be the host rock, to < −14 ‰ near the crack, a feature also found in the SIMS independent of microbially mediated processes. While ac- profile. This finding is congruent with the notion of hiatuses cording to Kadlec et al. (2008) and Filippov (2000), no being caused by permafrost build-up. δ18O in Siberian me- iron-bearing minerals are present in the host rock above the teoric water shows a strong relationship with temperature cave, it seems that early geological studies in the vicinity of (Brezgunov et al., 1998), and cooling associated with per- the cave found sparse sulfide-rich deposits above the cave mafrost development would likely lead to very negative δ18O (Odintsev et al., 1947; Egorov, 2011). The reason why no values just before a hiatus. Permafrost thaw with the onset of iron oxides have been recorded since then remains unknown. warmer periods again allows water infiltration, probably with Oxidation of pyrite in the host rock could be a potential isotopically depleted water entering the cave initially. source of iron by reaction with either oxygen or ferric iron Thus, while δ13C is likely affected by microbial activity (usually occurring in acidic environments) by acidophilic mi- under beneficial circumstances, this influence is of too small crobes, or under anoxic conditions at a circumneutral pH a scale to be recovered by conventional IRMS analysis. δ18O through the “thiosulfate pathway” (Luther and George, 1987; is more likely governed by abiotic processes such as temper- Moses et al., 1987; Schippers and Jorgensen, 2001). Ferric ature and the δ18O signature of the meteoric water, and no ions produced by the oxidation of solid or aqueous phase microbial influence can be attested in this case. Fe(II) with oxygen can then be precipitated and immobilized Speleothem calcite formation through purely abiogenic as a hydroxide, an oxide, a phosphate or a sulfate, or, if bound processes has already been challenged (Jones, 2009, 2010, to soluble organic ligands, converted to soluble complexes 2011), and it seems that repeated microbial-calcite associa- and dispersed from their source. However, no acidic condi- tion is found in a wide variety of climatic settings in caves tions (leading to carbonate dissolution) or anoxic deposits (e.g., Frisia et al., 2012; Jones, 2011). Further investigations (leading to OM accumulation) that could support this inter- combining microscopy and isotope geochemistry (SIMS) are pretation have been found on top of the cave. Botovskaya required in caves from different climatic zones to constrain Cave was within the vadose zone during stalactite deposition www.biogeosciences.net/10/6115/2013/ Biogeosciences, 10, 6115–6130, 2013 6126 M. Pacton et al.: The role of microorganisms in the formation of a stalactite in Botovskaya Cave, Siberia

(stalactite formation can only happen in air-filled voids), pre- the hypothetical peat bog into the Boty River valley, followed venting anoxic or acidic conditions in carbonate rock above by oxidation of the peat deposits, associated mobilization of the cave. Another reason to reject this source is that iron de- Fe and Mn, and formation of Mn-Fe deposits there. Disap- posits/coatings have only been observed in the oldest part pearance of the peat bog sediment would have stopped the of the stalactite, and have not been found in other young infiltration of Mn and Fe, and thus the recording of Mn-Fe (i.e., Holocene, MIS-5.5, MIS-7, MIS-9 and MIS-11) nearby deposits in the stalactite. speleothems from this cave. If the host rock were a source of iron, we would expect transport of iron into the cave during 5.6 Potential implications of microbes for speleothem all speleothem growth periods (warm interglacials without genesis continuous permafrost). Although we cannot strictly rule out the sulfide-rich de- The hypothesis that the growth of stalactites and stalagmites posit hypothesis at this time, the most likely hypothesis to can be mediated by microbes has already been postulated by permit Fe and Mn mobilization remains the presence of a many studies (e.g., Cox et al., 1989a, b; Cunningham et al., local peat bog above the cave at the time of deposition and 1995; Cañaveras et al., 2001; Jones, 2001, 2010; Melim et al., the seeping of acidic/anoxic water from there into the rock 2001, 2008, 2009; Basker et al., 2005, 2007, 2009; Frisia et fissures above the cave. al., 2012). However, recognizing and identifying the biosignatures of microbial processes is inherently difficult, espe- 5.5 Palaeoenvironmental appraisal of the deposited cially in fossil systems such as speleothems that lack pre- crusts served microbes and/or EPS (Barton et al., 2001; Barton and Northup, 2007; Engel, 2007; Melim et al., 2009; Jones et al., Siberian peats are especially rich in Fe and Mn (Efremova et 2011). To our knowledge this is the first study that constrains 13 al., 2001) and were widely distributed in this area during the δ Ccarb depletion along with detailed microscopical inves- Pleistocene and Holocene (e.g., Krivonogov et al., 2004). At tigations, thereby restricting a discussion of broader (geo- present, small peat bogs are common on the high flat plateau graphical) implications. Nevertheless, such records might oc- near the cave. Although no peat bog is found above this part cur in other climatic settings, thus opening up the possibil- of the cave today, it is possible that one existed there earlier, ity of investigating the role of microbes as contributors to causing the peat bog water, rich in Fe, Mn and organic mate- speleothem formation under temperate and tropical condi- rials, to infiltrate the cave, and was the most likely source for tions (e.g., Frisia et al., 2012; Jones et al., 2011), and pos- the Mn(II) and Fe(II) deposited as dark crusts. sibly as recorders of climatic changes. Although microbial Most of the iron present in the modern soil is of the param- carbonate formation can be mediated under different climatic agnetic Fe3+ form, which may be the result of weathering of conditions, including cold and dry climates especially re- paramagnetic minerals during pedogenetic processes (Evans lated to permafrost occurrence, e.g., Pellerin et al. (2009), and Heller, 2004; Kadlec et al., 2008). Enhanced erosion the timing of periods of calcite growth initiation and ces- might have occurred during the Middle or Late Pleistocene sation cannot be fixed with certainty at this stage. For ex- above the cave (Kadlec et al., 2008), but detailed chrono- ample, carbonate lamination in stromatolites is interpreted logical constrains are lacking. Precipitation of iron and man- as recording the periodic response of a microbial commu- ganese oxides in caves depends on the pH of the medium nity to daily, seasonal, or yearly environmental forcing, and and is closely tied to the distance from the substrate rock also to regional climate forcing (Petryshin et al., 2012), but (Gazquez et al., 2011). At around pH 6, precipitation of iron the timing of carbonate laminae formation can vary consid- oxides is frequent, whilst close to pH 8.5, manganese oxides erably depending on geological settings (e.g., Chivas et al., can be precipitated (Onac, 1996). However, biological iron 1990; Paull et al., 1992; Font et al., 2010; Petryshin et al., oxidation can also occur at circumneutral pH (Emerson and 2012). It has been suggested that microbial processes sim- Moyer, 1997). This is in agreement with the fact that iron ilar to stromatolite-like layers in speleothems mark periods oxides are found contemporaneous with EPS. Moreover, the of reduced drip rate, and thus might potentially be indicative calcitic microboring casts observed under the ferrous cover of dry phases (Frisia et al., 2012). Similarly, in this study, would have been dissolved at a lower pH. hiatuses caused by permafrost build-up are closely related to In the stalactite, the Mn-Fe mineralization is concentrated porous cracks (small low-magnesium calcite rhombs and lo- on the side where the layers’ thickness is reduced, indicating cal microbial filaments) and associated with 13C depletion. that Mn-Fe deposits precipitated in places where the water Therefore, these microbial biosignatures might be an indi- flow was minimal. Higher water flow took place on the other cator of environmental change (freezing or dry conditions) side of the stalactite, leading mainly to calcite deposition. It in different climatic settings. Similar investigations combin- is possible that microbial colonies preferred to occupy the ing microscopy and in situ isotope geochemistry (SIMS) are area with minimal water flow, as it is known that increasing definitely required in caves from different climatic zones in hydrodynamic conditions reduce biofilm adhesion (Lau and order to better constrain the role of microbes in speleothem Liu, 1993). Surface erosion eventually led to the draining of formation related to hiatuses and/or carbonate disruptions.

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6 Conclusions crusts into even older periods, from Middle Pleistocene to Pliocene. The mere presence of microbes in a speleothem does not inform us if they played a formative role in the growth and development of that speleothem. Stalactite formation Supplementary material related to this article is Botovskaya Cave (Siberia) is the result of a combination available online at http://www.biogeosciences.net/10/ of both biotic and abiotic processes. It is composed of an 6115/2013/bg-10-6115-2013-supplement.zip. alternation of columnar prismatic calcite and anhedral calcite locally associated with ferromanganese oxides. Anhedral calcite crystals are closely tied with permineralized EPS as Acknowledgements. We thank ETH Zurich, Pethros (Petrobras) and amorphous Mg-Si phases that constitute the base of each the Swiss National Science Foundation (grants No. 200020_127327 layer. The origin of ferromanganese deposits is related to and CRSI22_132646/1) for financial support, and the University the mobilization of polymetallic minerals in peats above the of Zurich for access to electron micrograph scanning facilities. 2+ 2+ cave. Metal ions (including Fe and Mn ) released into We thank G. M. Henderson from the University of Oxford for the cave under reducing conditions are oxidized and fixed laboratory and funding support. Birgit Plessen (GFZ Potsdam) by microbes and evolve further to oxides and hydroxides of kindly supported stable isotope analyses of stalagmites BOTI-881 low crystallinity through EPS. Both carbonate and ferroman- and BOTI-4537. We also thank Saskia Bindschedler, Sophie ganese oxides are triggered by passive mineralization of EPS Verheyden and Amy Frappier for helpful constructive comments (i.e., EPS-influenced organomineralization processes), sug- that greatly improved the manuscript. gesting that no metabolisms are required for mineral forma- Edited by: D. Gillikin tion in caves. Therefore, microbial involvement in the stalactite’s formation is supported by multiple evidence, including: References – internal fabrics similar to EPS morphology, Baldini, J., McDermott, F., Hoffmann, D., Richards, D., and Clip- – microbial permineralization as amMg-Si phases, and son, N.: Very high-frequency and seasonal cave atmosphere PCO variability: implications for stalagmite growth and oxy- 13 2 – consistent depletion of δ C values for porous (pu- gen isotope-based paleoclimate records, Earth Planet. Sci. Lett., tative biogenic) versus columnar (putative abiogenic) 272, 118–129, 2008. layers. 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